Alzheimer's disease (AD) is a progressive degenerative disease of the brain primarily associated with aging which results in loss of memory and orientation. As the disease progresses, motor, sensory and linguistic abilities are also affected until there is global impairment of multiple cognitive functions of the brain. These cognitive losses occur gradually, but typically lead to severe impairment and eventual death in the range of four to twelve years.
Alzheimer's disease is characterized by two major pathologic observations in the brain: neurofibrillary tangles and amyloid (or neuritic) plaques. Neurofibrillary tangles occur not only in Alzheimer's disease but also in other dementia-inducing disorders, while amyloid plaques are peculiar to AD. Smaller numbers of these lesions in a more restricted anatomical distribution are found in the brains of most aged humans who do not have clinical AD. Amyloidogenic plaques and vascular amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type (HCHWA-D). At present, a definitive diagnosis of AD usually requires observing the aforementioned lesions in the brain tissue of patients who have died with the disease or, rarely, in small biopsied samples of brain tissue taken during an invasive neurosurgical procedure.
Neurofibrillary tangles are characterized as networks of microtubules and microfilaments which were once structural supports running symmetrically through the nerve cells that transported nutrients but have degenerated into dysfunctional tangled masses. They can be described histologically as non-membrane bound bundles containing paired, helically wound filaments (PHF) that are approximately 10 nm in length and located in the perinuclear cytoplasm of certain neurons. Major components of paired helical filaments are highly phosphorylated tau proteins (PHF-tau) of 60 kDa, 64 kDa and 68 kDa. Aβ peptide is also a component of these tangles. Tau belongs to the family of microtubule-associated proteins and plays a role in the microtubule assembly and stabilization. In certain other neurodegenerative disorders, including corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and Pick's disease, hyperphosphorylated tau proteins also accumulate in brain tissue in association with abnormal filaments. Recent research indicates that the pattern of hyperphosphorylation and the resulting ultrastructure of the helical filaments are somewhat different in each type of disease.
Amyloid plaques, on the other hand, are peculiar to and a defining feature of AD. Amyloid plaques are predominantly composed of amyloid beta peptide (Aβ, also sometimes designated as βA4). Aβ is derived by proteolysis of the amyloid precursor protein (APP) and is comprised of 39–43 amino acids. Several proteases called secretases are involved in the processing of APP. It appears that the abnormal processing and deposition of Aβ in areas of the brain responsible for cognitive activities is a major factor in the development of AD. Cleavage of APP at the N-terminus of the Aβ peptide by β-secretase and the C-terminus by one or more γ-secretases constitutes the amyloidogenic pathway, i.e., the pathway by which Aβ peptide is formed. Cleavage of APP by α-secretase and the same or a different gamma secretase produces α-sAPP, a secreted form of APP that does not result in amyloid plaque formation. This alternate pathway precludes the formation of AB. It has been proposed that Aβ peptide accumulates as a result of the processing of APP by β-secretase and that therefore inhibition of the activity of this enzyme is desirable for treatment of AD. See for example, β-Amyloid and Treatment Opportunities for Alzheimer's Disease, Sabbagh, M., et al., Alz. Dis. Rev. 3, 1–19, (1997).
Several lines of evidence indicate that progressive cerebral deposition of particular amyloidogenic proteins, β-amyloid proteins, (Aβ), play a seminal role in the pathogenesis of AD and can precede cognitive symptoms by years or decades. See, Selkoe, Neuron 6:487 (1991). Aβ is released from neuronal cells grown in culture and is present in cerebrospinal fluid (CSF) of both normal individuals and AD patients. See, Seubert et al., Nature 359:325–327 (1992).
Although diffuse deposition of Aβ peptide occurs in most all humans with aging, the formation of amyloid plaques occur only in AD patients. Formation of these plaques is believed to occur over a period of years or even decades. The Aβ peptide in amyloid plaques is always folded in a particular three-dimensional pattern called a beta-pleated sheet and appears to be chemically modified as well, which could explain the association of the Aβ peptides into the larger, denser plaques, rather than the diffuse deposits normally seen. Associated with this central core of Aβ peptide in the amyloid plaque are surrounding abnormal neurites and several types of altered glial cells. Glial cells normally associate with neurons and perform support and protective functions. On the outside of the plaque are reactive astrocytes, which are a type of glial cell typically found in injured brain areas. Additionally many other biochemical components, including enzymes, proteoglycans and apolipoproteins are present in the plaques. For a discussion of the formation of these plaques see for example: Sabbagh, M., et al., cited supra.
The neurons touching the amyloid plaques are progressively debilitated and ultimately die. At present it is not known whether the Aβ peptide is neurotoxic in itself or if the secondary features of the amyloid plaques, e.g., the abnormal glial cells, cause the nerve cells to die. Researchers have demonstrated that the Aβ peptide has neurotoxic effects in vitro. Still other researchers have demonstrated that the 25–35 amino acid sequence of Aβ peptide is similar to that of substance P, an endogenous neuropeptide compound present in certain brain tissues and having neuroexcitatory effects. Co-administration of substance P in the study blocked the neurotoxic effect of Aβ peptide in rats. See: An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P. Kowall N W, et al., Proc Natl Acad Sci USA 88 (16) p7247–51 (1991). Another study reports that Aβ peptide is neurotoxic through its interference with Ca++ homeostasis. Korotzer A. R., et al., Differential regulation by beta-amyloid peptides of intracellular free Ca2+ concentration in cultured rat microglia. Eur. J. Pharmacol., 288 (2):125–30 1995. Further, some studies have proposed that Aβ peptide is responsible for the hyperphosphorylation of tau, a microtubule associated protein, which results in formation of PHFs and neurofibrillary tangles as described above. Thus, with Aβ peptide clearly linked to the formation of amyloid plaques and implicated in the formation of neurofibrillary tangles in AD, there is a need for agents and methods for reduction of Aβ peptide in vivo.
At present there are no published means for specifically inhibiting the β-secretase enzyme, or even structural identification of the β-secretase enzyme is or a peptide sequence of its active site. However, a commonly assigned application naming John Anderson, Guriqbal Basi, et al. as inventors and entitled: β-Secretase Enzyme Compositions and Methods, identifies the enzyme and methods of use thereof. Additionally, a commonly assigned application naming Varghese John, Jay Tung, Roy Hom and Larry Fang as inventors and entitled: Dipeptide Inhibitors of β-Secretase, describes and claims dipeptide inhibitors of the β-secretase enzyme. The two above-identified applications are being filed on the same day as the present application. The contents of these co-pending applications are hereby incorporated by reference in their entirety for all purposes. Additionally, U.S. Pat. No. 4,636,491 to Bock, et al. discloses certain tetrapeptides having renin inhibitory activity. Some of the compounds disclosed in the broadest Markush description by Bock encompass some of the compounds of the present invention. However, all the specific examples of Bock are directed to the sequence: Phe-His-Sta-Leu or to the same sequence containing derivatives of statine. The sequence is neither claimed, nor operative in the β-secretase inhibition disclosed herein. It is believed that the compounds claimed herein are patentable as a selected subgenus of the broad Markush disclosure of Bock.